Gene encoding HM1.24 antigen protein and promoter thereof

ABSTRACT

There are provided a genomic DNA comprising 4 exons encoding the amino acid sequence as set forth in SEQ ID NO: 2 and 3 introns ligating them, and a splicing variant of said genomic DNA; as well as a DNA having the base sequence as set forth in SEQ ID NO: 4 and a promoter activity and the fragment thereof, and uses thereof.

TECHNICAL FIELD

The present invention relates to a genomic gene encoding HM1.24 antigen protein, a promoter of the gene encoding HM1.24 antigen protein, and uses thereof.

BACKGROUND ART

Mouse anti-HM1.24 monoclonal antibody has been prepared using a human myeloma cell line KPC-32 as an immunogen (Goto, T. et al., Blood 84: 1922-1930, 1994). The HM1.24 antigen that is recognized by this antibody is a membrane protein having a molecular weight of 29 to 33 kDa that is overexpressed on the surface of myeloma cells. Furthermore, for normal cells its expression has been confirmed in immunoglobulin-producing B cells (plasma cells, lymphoplasmacitoide cells), but expression is rarely observed in the other cells and tissues (Goto T. et al., supra). However, nothing is known about HM1.24 antigen except its expression distribution and molecular weight.

According to the present invention, as a result of the cloning of genomic DNA encoding HM1.24 antigen, the determination of its nucleotide sequence and the deduced amino acid sequence, and further homology search, HM1.24 antigen was demonstrated to be a molecule identical with BST2 that is a surface antigen expressed on the stroma cells isolated from the bone marrow of patients with myeloma, and the bone marrow and the snynovial membrane of patients with rheumatoid arthritis. BST2 has been shown to have an ability of supporting the growth of pre-B cells and is thought to be involved in the pathology of rheumatoid arthritis, but its other physiological functions are not known (Ishikawa J. et al., Genomics 26: 527-534, 1995).

In the production of a useful gene product derived from an animal by means of genetic engineering, it often happens that the gene is not expressed, a gene product, protein, does not take a correct conformation, post-translational modification does not occur correctly and the like, when a microorganism host such as Escherichia coli, Bacillus subtilis, or yeast is used. In order to solve such problems, animal cells are often used as hosts, in which case, the selection of a promoter has a great impact on expression efficiency. Conventional, frequently used promoters for animal cells include SV40 promoter, cytomegalovirus promoter, actin promoter, and the like.

DISCLOSURE OF THE INVENTION

Considering the above state of art, the present invention provides a genomic DNA encoding HM1.24 antigen protein.

The present invention also provides a process for producing HM1.24 antigen protein using animal cells by means of said genomic DNA.

When a useful gene product is to be produced in large quantities using animal cells as a host, conventionally used promoters for animal cells are not always satisfactory in terms of transcription activity, and hence there is a great need for the development of stronger promoters. Thus, it is an object of the present invention to provide a DNA having a stronger promoter activity as a promoter for animal cells and uses thereof.

In order to solve the above problems, the present invention provides a genomic DNA encoding HM1.24 antigen protein, said DNA comprising 4 exon regions encoding the amino acid sequence as set forth in SEQ ID NO: 2. As an example of the above genomic DNA, the present invention provides a genomic DNA having 4 exons encoding the amino acid sequence and 3 introns as set forth in SEQ ID NO: 2.

The present invention also provides a splicing variant of the above genomic DNA. Specific examples include a splicing variant lacking exon 2, a splicing variant lacking exons 2 and 3, and the like.

The present invention also provides a process for producing HM1.24 antigen protein which method comprises culturing animal cells transformed with an expression vector comprising the above genomic DNA.

The present invention further provides a promoter sequence DNA having the nucleotide sequence of the 5′-non-coding region as set forth in SEQ ID NO: 4 or a DNA fragment of said sequence having a promoter activity in animal cells.

The present invention also provides a DNA that hybridizes with the above DNA or a fragment thereof under a stringent condition and that has a promoter activity in animal cells. The above DNA having promoter activity is preferably derived from animal cells, in particular mammalian cells.

The present invention also provides a DNA that has been modified by the deletion, addition and/or substitution with other nucleotides, of one or a plurality of nucleotides in the nucleotide sequence of the 5′-non-coding region as set forth in SEQ ID NO: 4 and that has a promoter activity in animal cells.

The present invention also provides a recombinant DNA wherein a useful gene is operably linked to the above DNA having a promoter activity. As the above useful genes, there can be mentioned, for example, nucleic acids selected from the group consisting of nucleic acids encoding useful proteins, antisense DNA, antisense RNA, nucleic acids encoding decoys, and ribozyme.

The present invention also provides a vector comprising the above recombinant DNA. The vector is a plasmid vector or a virus vector.

The present invention also provides animal cells into which the above recombinant DNA has been introduced.

The present invention also provides animal cells that have been transformed with the above vector.

The present invention also provides an animal having the above animal cells.

The present invention also provides a method of expressing a useful gene which method comprises culturing animal cells into which the above recombinant DNA has been introduced.

The present invention also a process for producing a useful protein which process comprises sulturing animal cells transformed with an expression vector comprising a nucleic acid encoding a useful protein operably linked to the above DNA having a promoter activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing that shows the nucleotide sequence (SEQ ID NO:1) of cDNA encoding HM1.24 antigen protein and the corresponding amino acid sequence (SEQ ID NO:3). The underlined part shows a N-type sugar chain binding motif.

FIG. 2 is a drawing that shows the nucleotide sequence (SEQ ID NO:1) of cDNA encoding HM1.24 antigen protein and the corresponding amino acid sequence (SEQ ID NO:3).

FIG. 3 is a schematic diagram showing a clone P3.19 isolated using the panning method and 5 clones (IS1 to IS5) isolated by the immunoscreening method.

FIG. 4 is a drawing that shows the result of flow cytometry analysis using anti-HM1.24 antibody (A: CHO/NEO, B: CHO/HM). The histogram of anti-HM1.24 antibody is shown by a solid line, and that of the control antibody (UPC10) that showed a matching isotype is shown by a broken line. In the figure, the abscissa refers to fluorescence intensity and the ordinate to cell count.

FIG. 5 is a photograph in which the expression of HM1.24 antigen in each cell line and HM1.24 antigen-expressing CHO cells was detected by the immunoprecipitation/Western blotting method using anti-HM1.24 antibody. After immunoprecipitation using the anti-HM1.24 antibody-bound Sepharose 4B (lanes 1-6) or unbound Sepharose 4B (lanes 7 and 8), Western blotting was carried out using anti-HM1.24 antibody to detect HM1.24 antigen (shown on the right). (*: anti-HM1.24 antibody H chain).

FIG. 6 is a drawing that shows a restriction map of the 5′-untranslated region comprising the promoter region of the HM1.24 antigen protein gene.

FIG. 7 is a drawing that shows the nucleotide sequence of the 5′-untranslated region comprising the promoter region of the HM1.24 antigen protein gene (SEQ ID NO:4). Each transcription factor binding motif has been underlined.

FIG. 8 is a drawing that shows the nucleotide sequence of the 5′-untranslated region (SEQ ID NO:4) comprising the promoter region of the HM1.24 antigen protein gene. Each transcription factor binding motif has been underlined, the TATA-like sequence has been boxed, the transcription initiation point is represented by an arrow, and the region encoding 7 amino acids at the N-terminal of the protein is represented by the one-letter code of amino acids (SEQ ID NO:21).

FIGS. 9A-9B, (A) shows the position of a primer corresponding to the genome encoding HM1.24 antigen protein, and (B) shows the base sequence of each primer.

FIG. 10 is a drawing that shows a restriction map of genomic DNA encoding HM1.24 antigen protein and the positions of the corresponding exons and introns.

FIG. 11 is a drawing that shows a restriction map of genomic DNA encoding HM1.24 antigen protein (SEQ ID NO:2) and the corresponding amino acid sequence (upstream side) (SEQ ID NO:3). The arrows shows the transcription initiation point and the underline shows the N-type sugar chain binding motif.

FIG. 12 is a drawing that shows the nucleotide sequence of genomic DNA encoding HM1.24 antigen protein (SEQ ID NO:2) and the corresponding amino acid sequence (downstream side) (SEQ ID NO:3). The double underline shows the poly A-addition signal.

FIG. 13 is a drawing that shows the nucleotide sequence of a splicing variant of human HM1.24 antigen protein (SEQ ID NO:19) and the corresponding amino acid sequence (SEQ ID NO:20). The underlined part shows where the amino acid sequence is different from that of human HM1.24 antigen protein.

FIG. 14 is a drawing that shows the nucleotide sequence of the genomic DNA of HM1.24 antigen protein (SEQ ID NO:33). A genome was present which has mutations, a→g at position 178, g→a at position 262, and t→c at position 323, as well as a deletion of one of 9 a's near position 360. The symbol “★” represents a transcription initiation point. There was also a genome in which 19 bp at positions 93-111 repeat in tandem. The symbol “→” shows the position at the sense primer.

FIG. 15 shows the nucleotide sequence of genomic DNA of HM1.24 antigen protein (SEQ ID NO:33) and the corresponding amino acid sequence (SEQ ID NO:3). there was also a genome in which 8 base pairs at positions 551-558 were deleted. The symbol “←” shows the position of the antisense primer.

FIG. 16 is a drawing that shows the nucleotide sequence of genomic DNA (intron site) of HM1.24 antigen protein (SEQ ID NO:33). The → shows the position of the sense primer.

FIG. 17 shows the nucleotide sequence of genomic DNA of HM1.24 antigen protein (SEQ ID NO:33) and the corresponding amino acid sequence (SEQ ID NO:3). The → shows the sense primer, and the ← indicates the antisense primer.

FIG. 18 shows the nucleotide sequence of genomic DNA of HM1.24 antigen protein (SEQ ID NO:33) and the corresponding amino acid sequence (SEQ ID NO:3). There also was a genome in which 3 out of 5 c's near position 2314 were deleted. The ← shows the position of the antisense primer.

EMBODIMENT FOR CARRYING OUT THE INVENTION

A genomic gene comprising a genomic DNA and a promoter region of human HM1.24 antigen can be easily amplified by a PCR method using suitable primers. Thus, a genomic DNA of human HM1.24 antigen can be amplified by designing a sense primer that hybridizes to the 5′-end of a genomic DNA sequence as set forth in SEQ ID NO: 2 and an antisense primer that hybridizes to the 3′-end, and then by performing a PCR reaction using a polymerase, such as AmpliTaq (Perkin Elmer), LA-Taq (Takara Shuzo), and the like, and using as a template human genomic DNA prepared according to a standard method. A PCR product can be directly inserted into a cloning vector such as pCRII (Invitrogen) or pGEM-T (Promega).

By introducing a restriction enzyme recognition site into a sense primer or an antisense primer, it can be inserted into a desired vector.

Genomic DNA that contains a promoter region of human HM1.24 antigen can also be amplified by the same method. Thus, a desired DNA fragment can be obtained by designing a sense primer that hybridizes to the 5′-end of the sequence as set forth in SEQ ID NO: 4 and an antisense primer that hybridizes to the 3′-end, and then by amplifying by a PCR reaction using human genomic DNA as a template.

A genomic DNA encoding HM1.24 antigen protein of the present invention comprises, as shown in FIG. 10, 4 exons and 3 introns linking them, and their specific nucleotide sequences and deduced amino acid sequences of the exon regions are as shown in FIGS. 11 and 12 (SEQ ID NO: 2). Thus, exon 1 encodes from amino acid Met at position 1 to amino acid Val at position 95; exon 2 encodes from amino acid Met at position 96 to amino acid Glu at position 117; exon 3 encodes from amino acid Glu at position 118 to amino acid Arg at position 138; and exon 4 encodes from amino acid Arg at position 139 to amino acid Gln at position 180.

The present invention also provides splicing variants of genomic DNA encoding HM1.24 antigen protein. Splicing variants are those in which at least one, i.e. 1 to 3, of exons 1 to 4 has been removed, for example exon 2 or 3, or both of them are removed.

The present invention also provides splicing variants of genomic DNA encoding HM1.24 antigen protein having a nucleotide sequence of DNA in which the codon corresponding to each amino acid of an exon is out of position because the nucleotide sequence in an exon was deleted due to splicing.

Since the splicing variants have the reading frames of different amino acid sequences, they have amino acid sequences different from that of HM1.24 antigen protein encoded by exons 1 to 4. As an example of such splicing variants, there can be mentioned a splicing variant having the nucleotide sequence and the amino acid sequence as set forth in SEQ ID NO: 17.

A genomic DNA encoding the HM1.24 antigen protein of the present invention can be obtained by cloning a cDNA that encodes HM1.24 antigen protein, then using this cDNA to design a primer oligonucleotide, which is amplified by the PCR method using genomic DNA as a template. In order to clone cDNA, animal cells expressing HM1.24 antigen, for example KPMM2 cells, are cultured, and from the cell culture total RNA is extracted according to a standard method and then mRNA is enriched.

According to the present invention, based on the above mRNA, cDNA is synthesized by a standard method, which is fractionated using a low-melting point agarose gel. Then cDNA having a size of 0.7 kbp or greater is inserted into an expression vector pCOS1 or λExCell vector to prepare a library A which is used for screening by direct expression cloning, i.e. panning, and library B which is used for immunoscreening.

For screening by the panning method, an expression plasmid that constitutes library A was introduced into COS-7 cells using electroporation. After culturing, attached cells were scraped off and were contacted to a panning plate coated with anti-HM1.24 antibody to allow the cells expressing HM1.24 to be attached to the plate. Then plasmid DNA was extracted from the cells attached to the plate, amplified in E. coli, and used for the subsequent panning. The panning procedure was repeated three times to select clones that express antigen reacting with anti-HM1.24 antibody, and one of the clones was designated as clone P3.19.

Sequencing revealed that clone P3.19 consists of 1,012 bp and contains an open reading frame encoding 180 amino acids. The nucleotide sequence of the cDNA insert in this clone P3.19 and the corresponding amino acid sequence are shown in FIG. 1 and SEQ ID NO: 1. The amino acid sequence alone is shown in SEQ ID NO: 3.

For immunoscreening, on the other hand, a phage constituting library B was cultured together with E. coli NM522 on an agar plate, the expression product was transferred to a nitrocellulose filter, and the filter was contacted to an anti-HM1.24 antibody solution. Anti-HM1.24 antibody that was bound to the filter via binding with the expression product was detected with labeled anti-mouse immunoglobulin (Ig) serum.

This produced 5 positive clones: IS-1 to IS-5. The nucleotide sequences of these cDNA inserts were determined and were compared to the nucleotide sequence of the cDNA of the above P3.19. The comparison revealed, as shown in FIG. 3, that any cDNA in clones IS-1 to IS-5 was part of the cDNA of P3.19 and the 5′-end has been deleted in P3.19.

Then, after P3.19 was introduced into CHO cells to transform the cells, flow cytometry was performed using anti-HM1.24 antibody. The result confirmed, as shown in FIG. 4, that HM1.24 antigen was expressed. Furthermore, as shown in FIG. 4, P3.19 was confirmed to encode HM1.24 antigen by immunoprecipitation as well.

Then, as shown in FIG. 9A, the cDNA sequence was divided into four regions, and primer pairs were designed as shown in FIG. 9B to amplify each part. The genome library prepared according to a standard method was PCR-amplified using the above each pair of primers, which were then ligated together to obtain a full-length genomic DNA.

The result is shown in FIGS. 11 and 12, and SEQ ID NO: 2. As can be seen, the genomic DNA encoding HM1.24 antigen protein has 4 exons and 3 introns linking them. These relationships are schematically shown in FIG. 10, which also shows a restriction map of genomic DNA.

The present invention also relates to a process for producing HM1.24 antigen protein which method comprises culturing animal cells transformed with an expression vector into which the above genomic DNA has been inserted. As animal cells for use in this process, various animal cells, for example, described below with respect to the promoters of the present invention may be used, and cell cultures of humans, mammals other than humans, insects and the like are preferred. For example, HeLa etc. are used as the cell culture of humans; CHO, COS, myeloma, BHK, Vero, etc. are mentioned as the cell cultures of mammals other than humans; and cell cultures of silkworm etc. are mentioned as the cell cultures of insects. As vectors for introducing DNA encoding the HM1.24 antigen protein of the present invention into animal cells, for example phage vectors such as M13 are used.

Culturing of animal cells for producing HM1.24 antigen protein can be performed according to a standard method, and the isolation of HM1.24 antigen protein from the culture can also be performed according to a standard method.

Hybridoma HM1.24 producing mouse anti-HM1.24 monoclonal antibody that specifically recognizes HM1.24 antigen protein has been internationally deposited under the provisions of the Budapest Treaty as FERM BP-5233 on Sep. 14, 1995 with the National Institute of Bioscience and Human technology, Agency of Industrial Science and Technology, of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki pref., Japan.

The promoter of the present invention and the uses thereof will now be explained below.

The word “promoter” as used herein includes, but is not limited to, a region that is located 20-30 base pairs upstream to the transcription initiation point (+1) and that includes a TATA box or a TATA box-like region responsible for directing RNA polymerase to start transcription at a correct position, and, in addition to this region, it may include regions that are required for proteins, other than RNA polymerase, to associate with for adjusting expression. When the term “promoter region” is used in the present invention, it means a region that includes the promoter as used herein.

The words “promoter activity” as used herein means an ability or a function of being ligated to a useful gene downstream to the promoter in a state that enables expression, so that when introduced into a host (animal cell) it can produce either intracellularly or extracellularly a gene product of the useful gene. In general, the presence of absence and/or intensity of a promoter is expressed as the promoter activity by ligating, downstream to the promoter, a gene (reporter gene) encoding an easily quantifiable protein in a state that enables expression, introducing it into a host, and then determining the amount expressed of the protein. Thus, when the expression of gene products of a useful gene were confirmed either intracellularly or extracellularly after the useful gene was ligated downstream to the promoter and introduced into a host, the promoter should have a promoter activity in the host into which the gene was introduced.

The words “animal cells” as used herein includes cells derived from humans, but they are not limited to them as long as the promoter of the present invention has a promoter activity in an animal cell. For example, there can be mentioned mammals other than humans (for example, mice, rats, rabbits, goat, pigs, cattle, horses, dogs, monkeys, and chimpanzees), birds (for example, chickens, turkeys, quails, ducks, and geese), reptiles (for example, snakes, crocodiles, and turtles), amphibians (for example, frogs, salamanders, and newts), fish (for example, scads, mackerel, sea bass, sea breams, sea perch, yellowtails, tuna, salmon, trout, carp, sweetfish, eel, soles, sharks, rays, and sturgeons).

The words “useful genes” as used herein includes, for example, nucleic acids encoding proteins that can be expressed in animal cells, antisense DNA or sense DNA of genes derived from animal cells, nucleic acids encoding decoys that have genes encoding the binding proteins of transcription factors derived from animal cells or sequences of the binding sites of transcription factors or similar sequences, and ribozymes that cleave mRNA derived from animal cells.

As nucleic acids encoding protein that can be expressed in animal cells include, but not limited to, those derived from animals, and as long as they can be expressed in animal cells, those derived from microorganisms such as bacteria, yeasts, actinomycetes, fungi, Ascomycetes, and Basidiomycetes, or those derived from living organisms such as plants and insects are also included in the useful genes mentioned in this specification.

The words “nucleic acids encoding decoys” as used herein means DNA that have genes encoding the binding proteins of transcription factors derived from animal cells or sequences of the binding sites of transcription factors or similar sequences, which are introduced as “decoys” into cells so as to suppress the action of the transcription factors. The word “ribozymes” as used herein means those that cleave mRNA of specific proteins, and that inhibits the translation of these specific proteins.

Ribozyme can be designed from gene sequences encoding specific proteins and, for a hammerhead type ribozyme, for example, the method as described in FEBS Letter, 228: 228-230 (1988) can be used. In addition to the hammerhead type ribozymes, any type of ribozymes including the hairpin type ribozymes, the delta type ribozymes, and the like that cleave mRNA of these specific proteins and that inhibit the translation of these specific proteins can be included in the ribozyme as used herein.

By ligating a useful gene downstream to a DNA fragment having the promoter activity of the present invention in a state that enables expression, the expression of the useful gene can be enhanced. Thus, useful genes are expressed in animal cells into which recombinant DNA comprising DNA having the promoter activity of the present invention and a useful gene ligated in a state that enables expression has been introduced with or without using a vector. As a vector, a plasmid vectors or a virus vectors is preferably used. When vectors are not used, DNA fragments can be introduced according to the methods described in the literature [Virology, 52: 456 (1973); Molecular and Cellular Biology, 7: 2745 (1987); Journal of the National Cancer Institute, 41: 351 (1968); EMBO Journal, 1: 841 (1982)].

Animal cells having such recombinant DNA fragments of the present invention and animals having such animal cells are also encompassed in the scope of the present invention. As useful genes whose expression can be enhanced according to the present invention, there can be mentioned, as described above, DNA encoding protein, antisense DNA, antisense RNA, polynucleotides encoding a decoy, nucleotide sequences functioning as a decoy, ribozymes, and the like. The present invention also discloses methods of producing proteins of interest, and methods of expressing useful genes using a DNA fragment having the promoter activity of the present invention.

Thus, a process for producing protein is also encompassed in the scope of the present invention wherein said process comprises ligating a nucleic acid encoding protein downstream to DNA having the promoter activity of the present invention in a state that enables expression, culturing an animal cell transformed with a vector containing the recombinant DNA thus obtained, and harvesting said protein from the culture. Similarly, a method of expressing a useful gene comprising ligating a useful gene downstream to DNA having the promoter activity of the present invention in a state that enables expression, introducing the recombinant DNA thus obtained into an animal cell, and culturing, or the method of expressing a useful gene using an animal cell said method comprising transforming an animal cell with a vector containing said recombinant DNA and culturing said animal cell is also encompassed in the scope of the present invention.

DNA having the promoter activity of the present invention is DNA having the nucleotide sequence shown in the 5′-end non-coding region as set forth in SEQ ID NO: 4 or a fragment thereof retaining a promoter activity. The 5′-end non-coding region means the nucleotide sequence up to position 2040 in SEQ ID NO: 4. It is known that a size of 5 nucleotides or greater is required to exhibit a promoter activity in animal cells. Thus, fragments of DNA having the promoter activity of the present invention have a size of at least 5 nucleotides or greater, preferably 30 nucleotides or greater, and more preferably 2000 nucleotides or greater.

The present invention also includes DNA that can hybridize with DNA having the nucleotide sequence as set forth in SEQ ID NO: 4 under a stringent condition and that has a promoter activity. The hybridizing DNA is for example a genomic DNA library derived from natural sources, for example mammals such as humans, mice, rats, and monkeys. As a stringent condition, there may be mentioned for example a low stringent condition. By way of example, a low stringent condition is a washing condition provided by 42° C. in 5×SSC, 0.1% sodium dodecyl sulfate, and 50% formamide. More preferably, a high stringent condition may be mentioned. By way of example, a high stringent condition is a washing condition provided by 60° C. in 0.1×SSC and 0.1% sodium dodecyl sulfate.

The present invention also includes a DNA fragment that has been modified by the deletion, addition, and/or substitution with other bases, of one or a plurality of nucleotides in the nucleotide sequence of the promoter as set forth in SEQ ID NO: 4 and that retains a promoter activity. The degree of modification is in the range of 70% homology to the nucleotide sequence as set forth in SEQ ID NO: 4, preferably a homology of 80% or greater, and more preferably a homology of 90% or greater.

“Homology” as used herein means the degree of identity of residues exhibited by two or more non-complementary nucleotide sequences or amino acid sequences (Gene Cloning 2nd edition, T. A. Brown, Chapman and Hall, 1990). Thus, a homology of 90% means that 90 residues or more out of 100 are identical in two or more sequences.

Now, a process for producing DNA having the promoter activity of the present invention, a fragment and a modified version thereof will be explained below. The DNA having the nucleotide sequence as set forth in SEQ ID NO: 4 was PCR-amplified using a primer AP1 (5′-GTAATACGACTCACTATAGGGC-3′) (SEQ ID NO: 5) corresponding to an adapter and the HM1 primer (sequence: 5′-ATC CCC GTC TTC CAT GGG CAC TCT GCA-3′ (SEQ ID NO: 6) corresponding to nucleotide Nos. 47-72 of cDNA clone P3.19 cloned by the above-mentioned panning method, and a human genomic DNA library as a template, and then, using the PCR-amplified product as a template, a nested PCR is performed using the AP2 primer (sequence: ACTATAGGGCACGCGTGGT) (SEQ ID NO: 7) and the HM2 primer (sequence: 5′-ATA GTC ATA CGA AGT AGA TGC CAT CCA G-3′ (SEQ ID NO: 8) corresponding to nucleotides 19-40 of clone P3.19 to subclone into a cloning vector pCRII (Invitrogen). By sequencing, DNA having the nucleotide sequence as set forth in SEQ ID NO: 4 was obtained.

A DNA fragment having a promoter can be obtained, for example, in the following manner. A method of digesting a DNA subcloned into the above cloning vector pCRII with restriction enzymes, ScaI, BamHI, PvuII, PstI, etc., a method using an ultrasonication treatment, a chemical synthesis by the phosphoramidite method, a method of preparation using a polymerase chain reaction method etc. can be used. For example, a desired DNA fragment can be easily prepared by preparing a primer as appropriate from the DNA sequence of SEQ ID NO: 4 and then performing a polymerase chain reaction.

A hybridization method that utilizes the nucleotide sequence of the promoter of the present invention may be used to obtain a promoter of the present invention from a gene derived from other cells. In this case, for example, the following method can be used. First, a chromosomal DNA obtained from a gene source of other cells is ligated into a plasmid or a phage vector and then introduced into a host according to a standard method to construct a library. The library is cultured on a plate, and colonies or plaques grown are transferred to a nitrocellulose or a nylon membrane, which is subjected to denaturation to immobilize DNA onto the membrane. The membrane is incubated in a solution containing a probe (as the probe, a DNA fragment as set forth in SEQ ID NO: 4 or a portion thereof) previously labeled with ³²P etc. to form a hybrid between the DNA on the membrane and the probe.

For example, a DNA-immobilized membrane is subjected to hybridization with a probe in a solution containing 6×SSC, 1% sodium dodecyl sulfate (SDS), 100 μg/ml salmon sperm DNA, 5×Denhardt's at 65° C. for 20 hours. After hybridization, non-specific adsorption is washed off, and autoradiography etc. is performed to identify clones that hybridized with the probe. The procedure is repeated until a single clone that formed a hybrid is obtained. Into a clone thus obtained, DNA encoding a desired promoter should be inserted.

The above promoter that has been modified by the deletion, addition, and/or substitution of nucleotides can be prepared by, for example, conventionally known methods such as site-directed mutagenesis, or a PCR method.

The gene obtained is sequenced for its nucleotide sequence, for example, in the following manner to confirm the gene obtained is a promoter of interest. For the determination of the nucleotide sequence, in the case of a clone obtained by hybridization, the transformant is cultured in a test tube if it is E. coli, and a plasmid is extracted therefrom according to a standard method. This is cleaved with a restriction enzyme to extract an inserted fragment, which is subcloned into M13 phage vector etc., and the nucleotide sequence is determined by the dideoxy method or the like.

When the transformant is a phage, essentially similar steps can be employed to determine the nucleotide sequence. Basic procedures from culturing to nucleotide sequence determination are carried out as described in, for example, Molecular Cloning: A laboratory Manual, Second edition, T. Maniatis, Chapter One, pp. 90-104, Cold Spring Harbor Laboratory, 1989.

Whether the obtained gene is a promoter of interest or not can be determined by comparing the determined nucleotide sequence with the promoter of the present invention and estimating from its homology. If the obtained gene is thought not to contain an entire promoter, a synthetic DNA primer is constructed based on the obtained gene, missing regions are amplified by PCR, and using the obtained gene fragment as a probe DNA libraries or cDNA libraries are screened so that the nucleotide sequence of the entire conding region of the promoter that hybridizes to the promoter of the present invention can be determined.

The method of expressing useful genes of the present invention is characterized in that a DNA fragment obtained by ligating, in a state that enables expression, a useful gene downstream to the promoter of the present invention thus obtained is introduced into an animal cell and the resulting cell is cultured. In order to ligate, in a state that enables expression, a useful gene downstream to the DNA fragment of the promoter of the present invention prepared as above, the DNA ligase method or the homopolymer method can be used.

If DNA ligase is used for ligating of the two, they are ligated by digesting with restriction enzymes and, if they have the same restriction enzyme site, then both the DNA fragments are mixed in a reaction buffer as described in Molecular Cloning: A laboratory Manual, Second edition, T. Maniatis et al. ed., Chapter One, pp. 62, Cold Spring Harbor Laboratory, 1989, and adding DNA ligase thereto or, if the they do not share the same restriction enzyme site, the ends are blunt-ended with T4 DNA polymerase (manufactured by Takara), and then treated with DNA ligase as described above.

On the other hand, when the homopolymer method is used, ligating is effected by attaching a poly G chain to the 3′-end of a vector linearized with a restriction enzyme using terminal deoxyribonucleotidyl transferase and dGTP, attaching similarly a poly C chain to the 3′-end of the insert DNA, and then annealing these poly G chain and poly C chain by, for example, the calcium chloride method for introduction into E. coli [Proc. Natl. Acad. Sci. U.S.A., 75: 3727 (1978)].

The useful genes of interest that can be used in the present invention includes, but not limited to, the interleukin 1-12 gene, the interferon α, β, γ genes, the tumor necrosis factor gene, the colony stimulating factor gene, the erythropoietin gene, the transforming growth factor-β gene, the immunoglobulin gene, the tissue plasminogen activator gene, the urokinase gene, the horseradish peroxidase gene, and the like.

There can be mentioned, for example, genes of superoxide dismutase, tumor necrosis factor, insulin, calcitonin, somatostatin, secretin, growth hormone, endorphine, viral protein, amylase, lipase, alcohol dehydrogenase, and the like.

The DNA fragment obtained as above in which the DNA fragment of the present invention and a useful gene is ligated can be integrated into an appropriate vector to obtain a plasmid for gene expression. Examples of such vectors include pTM [Nucleic Acids Research, 10: 6715 (1982)], cos202 [The EMBO Journal, 6: 355 (1987)], p91203 (B) [Science, 228: 810 (1985)], BCMGSNeo [Journal of Experimental Medicine, 172: 969 (1990)], and the like.

The plasmid thus obtained for gene expression can be introduced into a suitable host by the calcium phosphate method [Molecular and Cellular Biology, 7: 2745 (1987)], the electroporation method [Proc. Natl. Acad. Sci. U.S.A., 81: 7161 (1984)], the DEAE-dextran method [Methods in Nucleic Acids Research, page 283, Column et al., ed., CRC Press, issued in 1991], the liposome method [BioTechniques, 6: 682 (1989)], and the like.

Examples of such host cells include COS cells, HeLa cells, CHO cells, BHK-21 cells, and the like. By culturing the resulting transformed cells in a suitable medium, the useful gene product of interest can be obtained in an efficient manner.

EXAMPLES

The present invention will now be explained in more detail with reference to the following working Examples and Reference Examples.

Reference Example 1 Cloning of cDNA Encoding HM 1.24 Antigen Protein

1) Cell lines

Human multiple myeloma cell lines RPM18226 and U266 were cultured in a RPMI1640 medium (GIBCO-BRL) supplemented with 10% fetal bovine serum (FBS), and a human multiple myeloma cell line KPMM2 (Japanese Unexamined Patent Publication (Kokai) No. 7-236475) was cultured in a RPMI1640 medium (GIBCO-BRL) supplemented with 208 fetal bovine serum.

2) Construction of cDNA library

Total RNA was isolated from 1×108 KPMM2 cells by a guanidine thiocyanate/cesium chloride method, and mRNA was purified using the Fast Track mRNA Isolation Kit (Invitrogen). After synthesizing cDNA from 10 μg of mRNA using NotI/oligo-dT₁₈ primer (Time Saver cDNA Synthesis Kit; Pharmacia Biotech), an EcORI adapter was ligated thereto. A cDNA larger than 0.7 kbp was fractionated using 1.0% low-melting point agarose gel (Sigma), and digested with NotI. It was then inserted into the EcoRI/NotI site of a pCOS1 expression vector or a λExcell vector (Pharmacia Biotech) to prepare a library (library A) for use in direct expression cloning (screening by panning) and a library (library B) for use in immunoscreening, respectively.

The pCOS1 expression vector was constructed from HEF-PMh-gλ1 (see WO92-19759) by deleting the contained gene with EcoRI and SmaI digestion, and then by ligating the EcoRI-NotI-BamHI Adaptor (Takara Shuzo).

3) Panning

Library A was introduced into COS-7 cells by the electroporation method. Thus, 20 μg of a plasmid DNA (containing 5×10⁵ independent clones) was mixed with 0.8 ml of cells (1×10⁷ cells/ml in PBS), and the mixture was subjected to electroporation under a condition of 1.5 kV and 25 μFD capacity using the Gene Pulses (Bio-Rad). After being allowed to stand at room temperature for 10 minutes, the cells were suspended in a DMEM (manufactured by GIBCO-BRL) containing 108 FBS, divided into four 100 mm culture dishes, and cultured at 37° C. for 72 hours.

After culturing, the cells were washed with a phosphate saline buffer (PBS), and were scraped off by adding PBS containing 5 mM EDTA to adjust the cell suspension to 1 to 2-10⁶ cells/ml in PBS containing 5% FBS and 0.02% NaN₃. The cells were then allowed to stand on a panning plate (see below) coated with anti-HM1.24 antibody for 2 hours, and the plate was gently washed three times with 3 ml of PBS containing 5% FBS and 0.02NaN₃. After washing, plasmid DNA was recovered from the cells bound to the plate using Hirt's solution (Hitt J., Mol. Biol. 26: 365-369, 1983) (0.6% SDS, 10 mM EDTA). The recovered plasmid DNA was amplified in E. coli and used for the following panning.

A panning plate was prepared as follows. Three milliliters of an anti-HM1.24 antibody solution (10 μg/ml in 50 mM Tris-HCl, pH 9.5) was added to a cell culture dish (Falcon) with a diameter of 60 mm and was incubated at room temperature for 2 hours. After washing three times in 0.15 M NaCl, 3 ml of PBS containing 5% FBS, 1 mM EDTA, and 0.02% NaN₃ was added to the dish. After blocking by allowing to stand at room temperature for 2 hours, the panning plate was stored at −20° C. until use.

By repeating panning three times using a plasmid library (library A) containing 5-10⁵ clones as a starting material, a plasmid DNA having an about 0.9 kbp cDNA as an insert was concentrated. Using a Dye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems), the nucleotide sequence was determined using the 373A or 377DNA Sequencer (Applied Biosystems). The result revealed that clone P3.19 comprises 1,012 by cDNA and has an open reading frame (23-549) encoding 180 amino acids (FIGS. 1 and 2) (SEQ ID NO: 1). The amino acid sequence deduced from the cDNA had a structure characteristic to type II membrane proteins and had two N-type sugar chain binding sites.

4) Immunoscreening

Library B was subjected to immunoscreening using anti-HM1.24 antibody. Thus, a phage library containing 1.5×10⁵ independent clones was layered on agar together with E. coli NM522 (Pharmacia Biotech) and was cultured at 42° C. for 3.5 hours. After culturing, a nitrocellulose filter (Schleicher & Schuell) pretreated with 10 mM IPTG was layered on the plate, and was further cultured at 37° C. for 3 hours. After the filter was washed with 0.058 (v/v) Tween 20-added TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl), 1% (w/v) BSA-added TBS was added thereto, and was blocked by incubating at room temperature for 1 hour.

After blocking, an anti-HM1.24 antibody solution (a 10 μg/ml blocking buffer) was added, incubated at room temperature for 1 hour, and 5,000-fold diluted alkaline phosphatase-conjugated anti-mouse Ig antiserum (picoBlue Immunoscreening kit; Stratagene) was added, which was further incubated at room temperature for 1 hour. Spots that reacted with the antibody were allowed to develop color with a developing solution (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl₂) containing 0.3 mg/ml nitroblue tetrazolium and 0.15 mg/ml 5-bromo-4-chloro-3-indolyl phosphate.

By immunoscreening, five positive clones were isolated, all of which were consistent with the partial sequence of P3.19 (FIG. 3). Homology search of them revealed that P3.19 is identical with the DNA sequence of BST-2 (Ishikawa J. et al., Genomics, 26: 527-534, 1995) expressed on the bone marrow or synovial stromal cells. The same molecule was obtained from two types of screening, which strongly suggested that the membrane protein encoded by P3.19 is the HM1.24 antigen molecule.

E. coli having the plasmid pRS38-pUC19 in which DNA encoding a human protein having the same sequence as the above-mentioned human HM1.24 antigen protein has been inserted in between the Xbal sites of pUC vector was designated as Escherichia coli DH5α (pRS38-pUC19) and was internationally deposited under the provisions of the Budapest Treaty on Oct. 5, 1993 with the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki pref., Japan, under an accession No. FERM BP-4434.

5) FACS analysis

Furthermore, in order to confirm that the protein encoded by p3.19 indeed binds to anti-HM1.24, antibody, a CHO transformant cell line in which P3.19 was introduced was established. Thus, after the P3.19 clone was introduced into CHO cells by the electroporation method, it was cultured in the presence of 500 μg/ml G418 (GIBCO-BRL) to obtain a CHO cell line that expresses HM1.24 antigen.

The cultured cells (1×10⁶) were suspended to the FACS buffer (PBS (−)2% FCS/0.1% NaN₃), HM1.24 antibody was added thereto, which was reacted on ice for 30 minutes. After washing in the FACS buffer, it was resuspended in a GAM-FITC solution (25 μg/ml in the FACS buffer; Becton Dickinson), and was further reacted on ice for 30 minutes. After washing twice with the FACS buffer, it was resuspended in 600 μl of the FACS buffer for measurement by the FACScan (Becton Dickinson).

As a negative control, UPC10 was used.

As result of FACS analysis, CHO cells in which P3.19 was introduced were shown to react strongly with anti-HM1.24 antibody, whereas no binding was observed in CHO cells (CHO/NEO) in which the control expression vector was introduced (FIG. 17). It was confirmed therefore that the protein encoded by P3.19 binds to anti-HM1.24 antibody.

6) Immunoprecipitation

After washing the cells twice in PBS they were destructed by ultrasonication in the cell lysate buffer method (50 mM sodium borate, 150 mM NaCl, 0.5% sodium deoxycholate, 18 Nonidet P-40, 0.1 mg/ml phenylmethylsulfonyl fluoride, protease inhibitor cocktail [Boehringer Mannheim]) to obtain a soluble fraction. The soluble fraction was added to anti-HM1.24 antibody-conjugated Sepharose 48 beads. After centrifugation, the precipitate was separated on SDS-PAGE (12% gel), which was transferred onto a PVDF membrane. The PVDF membrane was reacted with anti-HM1.24 antibody, and then with POD-anti-mouse IgG, and detected using the ECL kit (Amersham).

Each of myeloma cell lines KPMM2, RPMI8226, and U266 strongly expressed HM1.24 antigen, and the immunoprecipitation of the cell lysates thereof with anti-HM1.24 antibody allowed the specific detection of protein with a molecular weight of about 29 to 33 kDa (FIG. 5). In a similar experiment for CHO cell lines (CHO/HM) in which P3.19 was introduced, immunoprecipitants were confirmed in the CHO/HM cells as for the myeloma cell lines (FIG. 5, lane 4). Such immunoprecipitants could not be observed in the control cells (CHO/NEO) in which the expression vector pCOS1 was only introduced (FIG. 5, lane 5).

P3.19 encodes a protein having an estimated molecular weight of 19.8 kDa comprising 180 amino acids (FIG. 1) and has two N-type sugar chain binding motifs (FIG. 1). Thus, it suggested that the presence of substances having different molecular weights observed by immunoprecipitation was due to differences in the modification of N-type sugar chains. In fact, the immunoprecipitants were confirmed to bind to several lectins.

Reference Example 2 Preparation of Hybridomas that Produce Mouse Anti-HM1.24 Monoclonal Antibody

In accordance with the method of Goto, T. et al., Blood (1994) 84, 1992-1930, hybridomas that produce mouse anti-HM1.24 monoclonal antibody were prepared.

A plasma cell line KPC-32 (1×10⁷) derived from the bone marrow of a human patient with multiple myeloma (Goto, T. et al., Jpn. J. Clin. Hematol. (1941) 32, 1400) was injected to the abdominal cavity of a BALB/c mouse (bred by Charles River) twice every six weeks.

Three days prior to sacrificing the animal, 1.5×10⁶ KPC-32 was injected to the spleen of the mouse in order to further enhance the antibody-producing ability of the mouse (Goto, T. et al., Tokushima J. Exp. Med. (1990) 37, 89). After sacrificing the animal, the spleen was extracted and the extracted organ was subjected to cell fusion with the myeloma cell SP2/0 according to the method of Groth, de St. & Schreidegger (Cancer Research (1981) 41, 3465).

By Cell ELISA (Posner, M. R. et al., J. Immunol. Methods (1982) 48, 23) using KPC-32, a culture supernatant of a hybridoma was screened for antibody. 5×10⁴ KPC-32 was suspended in 50 ml of PBS and then was aliquoted to a 96-well plate (U-bottomed, Corning, manufactured by Iwaki), which was then air-dried at 37° C. overnight. After blocking with PBS containing 1% bovine serum albumin (BSA), the culture supernatant of the hybridoma was added thereto and incubated at 4° C. for 2 hours. Then, peroxidase-labeled anti-mouse IgG goat antibody (manufactured by Zymed) was reacted at 4° C. for 1 hour. After washing, o-phenylene diamine solution substrate solution (manufactured by Sumitomo Bakelite) was reacted at room temperature for 30 minutes.

Reaction was stopped by adding 2 N sulfuric acid and the absorbance was measured at 492 nm using the ELISA reader (manufactured by Bio-Rad). In order to remove the hybridomas that produce antibodies against human immunoglobulin, a culture supernatant of positive hybridomas had previously been adsorbed to human serum and the reactivity to other cell lines was screened by ELISA. Positive hybridomas were selected, and their reactivity to various cells was investigated by flow cytometry. The last selected hybridoma clone was cloned twice and injected to the abdominal cavity of a pristane-treated BALB/c mouse. The ascites was obtained from the mouse.

Monoclonal antibody was purified from the ascites of the mouse by ammonium sulfate precipitation and a Protein A affinity chromatography kit (Ampure Pa., manufactured by Amersham). The purified antibody was labeled with FITC using the Quick Tag FITC biding kit (manufactured by Boehringer Mannheim).

As a result, monoclonal antibodies produced by 30 hybridoma clones reacted with KPC-32 and RPMI 8226. After cloning, the reactivity of the culture supernatants of these hybridomas with other cell lines or peripheral blood mononuclear cells was investigated.

Of them, 3 clones were monoclonal antibodies that specifically reacted with the plasma cell. From the 3 clones, a hybridoma clone that was most useful for flow cytometry analysis and had a CDC activity to RPMI 8226 was selected and designated as HM1.24. The subclass of the monoclonal antibody produced by this hybridoma was determined by an ELISA using a subclass-specific anti-mouse rabbit antibody (manufactured by Zymed). Anti-HM1.24 antibody had a subclass of IgG2a k. The hybridoma HM1.24 that produces anti-HM1.24 antibody was internationally deposited under the provisions of the Budapest Treaty as FERM BP-5233 on Sep. 14, 1995 with the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki pref., Japan.

Example 1 Cloning of the Promoter Region of HM1.24 Antigen Gene

Since HM1.24 antigen was strongly expressed in all the myeloma cells analyzed so far, it is very likely that the expression of HM1.24 antigen is deeply involved in physiological characteristics of multiple myeloma. Thus, the elucidation of the mechanism of HM1.24 antigen expression is an important challenge, and the inventors have clarified the gene structure of the promoter region.

The promoter region of the HM1.24 antigen gene was isolated using the PromoterFinder DNA Walking kit (Clontech). From the nucleotide sequence of the 5′-end of clone P3.19 isolated by Panning, two PCR primers were designed: HM1 (5′-ATC CCC GTC TTC CAT GGG CAC TCT GCA −3′) (SEQ ID NO: 6) and HM2 (5′-ATA GTC ATA CGA AGT AGA TGC CAT CCA G −3′) (SEQ ID NO: 8). The first PCR was performed using primer API (attached to the kit) corresponding to the adapter and the HM1 primer according to the instruction manual attached to the kit, and then the PCR product was subjected to a nested PCR using the AP2 primer (attached to the kit) and the HM2 primer. After the final PCR product was purified, it was subcloned into the pCRII cloning vector (Invitrogen).

The promoter region gene was simply isolated by the. PCR method. Thus, PCR products of about 2.0 kb, 0.7 kb, and 0.3 kb were specifically amplified from the EcoRV, PvuII, and DraI libraries (Promoter Finder Kit; Clontech), respectively. They were demonstrated to be derived from the same genomic DNA based on the cleavage patterns with restriction enzymes (FIG. 6). As a result of sequencing the nucleotide sequences, gene sequence of cDNA from 5′-end to 1959 by upstream was determined (FIGS. 7 and 8) (SEQ ID NO: 4). By a binding motif search of known transcription factors, the presence of transcription controlling elements of AP-2, Sp1, NF-IL6, NF-kB, STAT3 or ISGF3, and the like was observed, suggesting the possibility that the expression is controlled by the stimulation by inflammatory cytokeines such as IL-6 or IFN-α.

IL-6 is known to serve as a growth factor of myeloma cells, and therefore it was strongly suggested that NF-IL6 and STAT3 that are transcription factors acting downstream of IL-6 are involved in the expression control of HM1.24 antigen in myeloma cells (FIGS. 7 and 8). The transcription initiation point was estimated nucleotide on the nucleotide sequence of PCR products amplified using the CapSwitch oligonucleotide (CapFnder Kit; Clonetech), and at 27 positions upstream thereof a TATA box-like sequence (TAATAAA) was observed (FIGS. 7 and 8).

Example 2 Cloning of Genomic DNA for HM1.24 Antigen

Genomic DNA for HM1.24 antigen was amplified from human genomic DNA (Clontech) prepared from a human genomic DNA library (Promoter Finder DNA walking kit; Clontech) or peripheral blood using each PCR primer shown in FIG. 9. After purification, PCR products were each subcloned into the pCRII vector and the nucleotide sequence was determined.

Genomic DNA encoding HM1.24 antigen was divided into four fragments, which were amplified from human genomic DNA prepared from a human genomic DNA library (Promoter Finder kit; Clontech) or human peripheral blood (FIG. 9). After confirming their nucleotide sequences, they were compared to the nucleotide sequence of HM1.24 antigen cDNA with a result that the HM1.24 antigen gene is composed of four exons and three introns of 850 bp, 183 bp, and 307 by (FIG. 10).

However, from the human genomic DNA library prepared from the human placenta tissue, a gene consisting of 3 exons lacking intron 3 was only amplified, suggesting the presence of a genomic gene having a different exon/intron structure. In any structure, two N-type sugar chain binding sites and three cysteine residues present in the extracellular region of HM1.24 antigen were all present in exon I (FIGS. 11 and 12) (SEQ ID NO: 2).

Example 3 Confirmation of HM1.24 Antigen Splicing Variants

In order to confirm the presence of splicing variants of HM1.24 antigen, HM1.24 antigen cDNA was amplified by the PCR method using as a template cDNA prepared from a human myeloma cell line KPMM2 according to the method described above. The sense primer BST2-N (SEQ ID NO: 17; ATG GCA TCT ACT TCG TAT GAC) used in the PCR corresponds to bases 10 to 30 of P3.19 (SEQ ID NO: 1) isolated herein, and the antisense primer S3 (SEQ ID NO: 18; AAC CGT GTT GCC CCA TGA) corresponds to bases 641-658 of P3.19.

PCR-amplified products were subcloned into a cloning vector pCRII (Invtrogen) and from the resulting independent clones, plasmid DNA was recovered, with a result that two inserts having different sizes of about 650 by and about 550 by were observed. After the determination of each nucleotide sequence, it was found that the insert of about 650 by had the sequence identical with that of P3.19 whereas the insert of about 550 by had a deletion corresponding to bases 294 to 422 of P3.19 (SEQ ID NO: 19). The region in which the deletion was observed corresponds to exons 2 and 3 of human HM1.24 antigen genomic DNA, indicating the presence of variants due to different splicing.

Example 4 Analysis of Polymorphism of HM1.24 Gene

In connection with polymorphism found in the HM1.24 gene, its relationship to multiple myeloma was investigated. The peripheral blood samples of normal healthy humans were supplied as the buffy coat of donated blood samples from Japan Red Cross Tokushima Blood Center. For patients with myeloma, the peripheral blood or bone marrow fluid was collected from patients in Tokushima University First Internal Medicine Hospital or affiliated hospitals. Blood samples were subjected to the Ficoll-Conrey density centrifugation to separate mononuclear cells. Myeloma cell lines were cultured in a RPMI1640 medium (GIBCO-BRL, Rockville, Md., U.S.A.) containing 10% fetal bovine serum at 37° C. in a 5% CO₂ incubator. The peripheral blood mononuclear cells or the myeloma cell lines were treated with the DNAZol reagent (GIBCO-BRL) according to the protocol to extract genomic DNA from the cells.

The nucleotide sequence was determined by the PCR-direct sequencing method. 5′-promoter region was amplified by PCR (30 cycles of 94° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 1 minute) with ampliTaq DNA polymerase (Perkin Elmer, Chiba) using primer 6S (TCCATAGTCCCCTCGGTGG) (SEQ ID NO: 22) and BST2B (ATAGTCATACGAAGTAGATGCCATCCAG) (SEQ ID NO: 23). The HM coding region was amplified by PCR with LA Taq DNA Polymerase (Takara Shuzo, Otsu) using primer HMP2K (AAAGGTACCAGCTGTCTTTCTGTCTGTC) (SEQ ID NO: 24) and BST2-R4 (GTGCTCTCCCCGCTAACC) (SEQ ID NO: 25). With the reaction mixture as a template, PCR was further performed with Ex Taq DNA polymerase (Takara Shuzo) using primer 8S (GGACGTTTCCTATGCTAA) (SEQ ID NO: 26) and BST2-R1 (AAAGCGGCCGCTCATCACTGCAGCAGAGCGCTGAG) (SEQ ID NO: 27).

The reaction mixture was purified by the QIA Quick PCR Purification Kit (QIAGEN, Tokyo), and reacted with the resulting PCR fragment as a template, using, as a primer, 6S or BST2B for the 5′-promoter region, and 8S, HMINTIF (AGGGGAACTCACCAGACC) (SEQ ID NO: 28), HMEX2F (ATGGCCCTAATGGCTTCC) (SEQ ID NO: 29), HMEX3F; (CATTAAACCATAAGCTTCAGG) (SEQ ID NO: 30), HMEX2R (CCCTCAAGCTCCTCCACT) (SEQ ID NO: 31), or BST2-R1 for the HM coding region by the BigDye Terminator Cycle Sequencing Kit (Perkin Elmer). The nucleotide sequence was determined using the ABI3777 DNA Sequencer (Perkin Elmer). The frequency of 8 base pair deletion in the vicinity of 20 base pairs upstream to the initiation codon of the HM1.24 gene was detected by PCR. Thus, PCR (30 cycles of 94° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 1 minute) was performed with ampliTaq DNA polymerase (Perkin Elmer, Chiba) using primers 8S and HST-R3 (GACGGATCCTAAAGCTTACAGCGCTTATC) (SEQ ID NO: 32). The reaction mixture was electrophoresed on a 4% agarose gel and detected by ethidium bromide staining.

Polymorphism of the 5′-promoter Region of the HM1.24 Gene

For samples from normal healthy humans and patients, the nucleotide sequence of the 5′-promoter region of the HM1.24 gene was determined. The result is shown in FIGS. 14-18 (SEQ ID NO: 33). There were samples for which nucleotide substitution in the underlined 187, 262, and 323 in FIG. 14, and deletions near 360 in FIG. 14 and near 555 in FIG. 15 were observed, and there was a sample for which the region of 366 to 558 could not be decoded. When the sequence described in FIGS. 14 to 18 (SEQ ID NO: 33) was termed as type A, and the mutation type having the above nucleotide substitution/deletion as type B, the sample for which the region of 366 to 558 could not be decoded is thought to be a heterozygote (AB) of A and B, and the sample that could be decoded as type A or B is thought to be a homozygote (AA) of A or a homozygote (BB) of B. In addition to the above polymorphism, it was clarified that 19 by was inserted at tandem at the double-underlined region in a myeloma cell line HS-sultan (type M).

Polymorphism of the HM1.24 Gene

For the genomic gene region of cell lines of type AA (U266, HS-sultan) and two samples of type BB from normal healthy humans, the nucleotide sequence was determined. As a result, it was found that 3 bases of c were missing near 2315 of intron 3 in the sequence of type B, whereas no mutation was observed in the coding region.

Gene frequency of Type A and Type B

Polymorphism and disease sensitivity were investigated. When 8-base deletion was detected by PCR near 20 base pairs upstream to the initiation codon of the HM1.24 gene, there was no difference in frequency of polymorphism between 94 cases of normal healthy humans and 46 cases of patients with myeloma (Table 1). In both the normal healthy humans and the patients with myeloma, type A gene was dominant as the gene distribution was about A:B=2:1. For cell lines, there was a bias to type A with 9 cases out of 11 being type AA.

No relationships were observed between the polymorphism of the HM1.24 promoter region and sensitivity to myeloma diseases.

TABLE 1 Frequency of polymorphism AA AB BB Total Normal healthy humans 43 37 14 94 (%) 45.7 39.4 14.9 Myeloma patients 21 21 4 46 (%) 45.7 45.7 8.7 Myeloma cell lines 9 2 0 (%) 81.8 18.2 0

Industrial Applicability

According to the present invention, there was obtained the genomic gene of HM1.24 antigen that is highly expressed in all myeloma cells. The genomic gene that encodes HM1.24 antigen is useful for analysis of HM1.24 antigen. Since HM1.24 antigen is strongly expressed, the promoter region is thought to have a strong promoter activity, and accordingly is useful for the expression of useful genes.

Reference to the microorganisms deposited under the Patent Cooperation Treaty, Rule 13-2, and the name of the Depository organ

Depository Organ

Name: the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology

Address: 1-3, Higashi 1-chome, Tsukuba city, Ibaraki pref., Japan

Organism (1)

Name: Escherichia coli DH5α (pRS38-pUC19)

Accession number: FERM BP-4434

Date deposited: Oct. 5, 1993

Organism (2)

Name: Mouse-mouse hybridoma HM1.24

Accession number: FERM BP-5233

Date deposited: Sep. 14, 1995

33 1 1014 DNA Mus sp. CDS (23)..(562) 1 gaattcggca cgagggatct gg atg gca tct act tcg tat gac tat tgc aga 52 Met Ala Ser Thr Ser Tyr Asp Tyr Cys Arg 1 5 10 gtg ccc atg gaa gac ggg gat aag cgc tgt aag ctt ctg ctg ggg ata 100 Val Pro Met Glu Asp Gly Asp Lys Arg Cys Lys Leu Leu Leu Gly Ile 15 20 25 gga att ctg gtg ctc ctg atc atc gtg att ctg ggg gtg ccc ttg att 148 Gly Ile Leu Val Leu Leu Ile Ile Val Ile Leu Gly Val Pro Leu Ile 30 35 40 atc ttc acc atc aag gcc aac agc gag gcc tgc cgg gac ggc ctt cgg 196 Ile Phe Thr Ile Lys Ala Asn Ser Glu Ala Cys Arg Asp Gly Leu Arg 45 50 55 gca gtg atg gag tgt cgc aat gtc acc cat ctc ctg caa caa gag ctg 244 Ala Val Met Glu Cys Arg Asn Val Thr His Leu Leu Gln Gln Glu Leu 60 65 70 acc gag gcc cag aag ggc ttt cag gat gtg gag gcc cag gcc gcc acc 292 Thr Glu Ala Gln Lys Gly Phe Gln Asp Val Glu Ala Gln Ala Ala Thr 75 80 85 90 tgc aac cac act gtg atg gcc cta atg gct tcc ctg gat gca gag aag 340 Cys Asn His Thr Val Met Ala Leu Met Ala Ser Leu Asp Ala Glu Lys 95 100 105 gcc caa gga caa aag aaa gtg gag gag ctt gag gga gag atc act aca 388 Ala Gln Gly Gln Lys Lys Val Glu Glu Leu Glu Gly Glu Ile Thr Thr 110 115 120 tta aac cat aag ctt cag gac gcg tct gca gag gtg gag cga ctg aga 436 Leu Asn His Lys Leu Gln Asp Ala Ser Ala Glu Val Glu Arg Leu Arg 125 130 135 aga gaa aac cag gtc tta agc gtg aga atc gcg gac aag aag tac tac 484 Arg Glu Asn Gln Val Leu Ser Val Arg Ile Ala Asp Lys Lys Tyr Tyr 140 145 150 ccc agc tcc cag gac tcc agc tcc gct gcg gcg ccc cag ctg ctg att 532 Pro Ser Ser Gln Asp Ser Ser Ser Ala Ala Ala Pro Gln Leu Leu Ile 155 160 165 170 gtg ctg ctg ggc ctc agc gct ctg ctg cag tgagatccca ggaagctggc 582 Val Leu Leu Gly Leu Ser Ala Leu Leu Gln 175 180 acatcttgga aggtccgtcc tgctcggctt ttcgcttgaa cattcccttg atctcatcag 642 ttctgagcgg gtcatggggc aacacggtta gcggggagag cacggggtag ccggagaagg 702 gcctctggag caggtctgga ggggccatgg ggcagtcctg ggtgtgggga cacagtcggg 762 ttgacccagg gctgtctccc tccagagcct ccctccggac aatgagtccc ccctcttgtc 822 tcccaccctg agattgggca tggggtgcgg tgtggggggc atgtgctgcc tgttgttatg 882 ggtttttttt gcgggggggg ttgctttttt ctggggtctt tgagctccaa aaaaataaac 942 acttcctttg agggagagca caccttaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaattc 1002 gggcggccgc ca 1014 2 2337 DNA Mus sp. 2 ccctccccta actccaggcc agactccttt cagctaaagg ggagatctgg atggcatcta 60 cttcgtatga ctattgcaga gtgcccatgg aagacgggga taagcgctgt aagcttctgc 120 tggggatagg aattctggtg ctcctgatca tcgtgattct gggggtgccc ttgattatct 180 tcaccatcaa ggccaacagc gaggcctgcc gggacggcct tcgggcagtg atggagtgtc 240 gcaatgtcac ccatctcctg caacaagagc tgaccgaggc ccagaagggc tttcaggatg 300 tggaggccca ggccgccacc tgcaaccaca ctgtggtaag ctcctcaact cctttggatg 360 gcctagtact aggcggtggg agggacaaga atctctcccc agaaatctga cccagggtgg 420 gtctccaggg agatgcaggg gaggtcctga aactgctcct gggcccccac atcaagggac 480 ctaggttccc ctaccagggt ttgtgggccc ctaacccagt ccagggcact ggtgtagggg 540 cagggtgtta aaactctcca gatcccccaa atcggggacc tcagtatccc cctgggactt 600 aggtgaattt ataaattctt tccagggcac tggtgtcggg ggccttgaaa ctcctcgtgg 660 gcaccagtcc tgggggagta gaaatcccta ttcagggttg aagggggacc tcaccagacc 720 ctgaaaaagg gggcttttga aattttcact tcatccctaa gaaactgaaa tattcacctg 780 ggtcctgata tgggggatct tgaaactctc gctgggcatg tcacttgggc ggggaaatcc 840 cactgcattc tggatttggt agggccctct aacttttctt gggccattgc tcaggcaatc 900 tggaaatgtc cactaaactt tggttatcga tagcctccaa gtttccacgt ggggtggcct 960 caaaactccc attttgagga cccacatgct tatgggtggc cctgggagag tgtgtggttg 1020 tggctgttct ttaaggttgg agaccatggt gcagagaggg ttggaagaaa acctgaaagg 1080 ggtttgcatt taagcccctc tgtccccagg acctagggag gaggcccagg tcccaggggc 1140 agcagccaaa ctccccaggc caaaacccca gattctaact cttcttagat ggccctaatg 1200 gcttccctgg atgcagagaa ggcccaagga caaaagaaag tggaggagct tgagggtgag 1260 aaagggagaa gggagagggc cggggagggg tgagtcaggt atggaagagg gggtgggggc 1320 agggagacca gggctggagg ttggggtaag ggggaggttc tgtcccagag tggagcaggg 1380 ccccagcatg gccacatgct gacccgcccc ctgtttctgt cctcccaccc taccaggaga 1440 gatcactaca ttaaaccata agcttcagga cgcgtctgca gaggtggagc gactgaggtc 1500 agagatagcc ttcccccgct accctccacc tgccacattc ctctcacccc cacatcccta 1560 gcccaagacc caggatctcc tttgctccca aaatccccat tgccccaagg gataaagttt 1620 gagtcccaca aaaggataac ttagccccta gggtcacaga gccatgggtg gccgctgtcc 1680 attccctccc cggtgacttg gattggggcg gtgcgggggg aactcccggg ggcggtgggc 1740 ttacagggag ggcggcagga gccaggacga gcagatgcct gatttgcccc catctgtacc 1800 gcagaagaga aaaccaggtc ttaagcgtga gaatcgcgga caagaagtac taccccagct 1860 cccaggactc cagctccgct gcggcgcccc agctgctgat tgtgctgctg ggcctcagcg 1920 ctctgctgca gtgagatccc aggaagctgg cacatcttgg aaggtccgtc ctgctcggct 1980 tttcgcttga acattccctt gatctcatca gttctgagcg ggtcatgggg caacacggtt 2040 agcggggaga gcacggggta gccggagaag ggcctctgga gcaggtctgg aggggccatg 2100 gggcagtcct gggtgtgggg acacagtcgg gttgacccag ggctgtctcc ctccagagcc 2160 tccctccgga caatgagtcc cccctcttgt ctcccaccct gagattgggc atggggtgcg 2220 gtgtgggggg catgtgctgc ctgttgttat gggttttttt tgcggggggg gttgcttttt 2280 tctggggtct ttgagctcca aaaaaataaa cacttccttt gagggagagc acacctt 2337 3 180 PRT Mus sp. 3 Met Ala Ser Thr Ser Tyr Asp Tyr Cys Arg Val Pro Met Glu Asp Gly 1 5 10 15 Asp Lys Arg Cys Lys Leu Leu Leu Gly Ile Gly Ile Leu Val Leu Leu 20 25 30 Ile Ile Val Ile Leu Gly Val Pro Leu Ile Ile Phe Thr Ile Lys Ala 35 40 45 Asn Ser Glu Ala Cys Arg Asp Gly Leu Arg Ala Val Met Glu Cys Arg 50 55 60 Asn Val Thr His Leu Leu Gln Gln Glu Leu Thr Glu Ala Gln Lys Gly 65 70 75 80 Phe Gln Asp Val Glu Ala Gln Ala Ala Thr Cys Asn His Thr Val Met 85 90 95 Ala Leu Met Ala Ser Leu Asp Ala Glu Lys Ala Gln Gly Gln Lys Lys 100 105 110 Val Glu Glu Leu Glu Gly Glu Ile Thr Thr Leu Asn His Lys Leu Gln 115 120 125 Asp Ala Ser Ala Glu Val Glu Arg Leu Arg Arg Glu Asn Gln Val Leu 130 135 140 Ser Val Arg Ile Ala Asp Lys Lys Tyr Tyr Pro Ser Ser Gln Asp Ser 145 150 155 160 Ser Ser Ala Ala Ala Pro Gln Leu Leu Ile Val Leu Leu Gly Leu Ser 165 170 175 Ala Leu Leu Gln 180 4 2061 DNA Mus sp. 4 actaaaagtc tctgatatgc agaaataatg gcataagctg tctttctgtc tgtcccctct 60 ctctctctct gcctcggctg ccaggcaggg aagggccccc tgtccagtgg acacgtgacc 120 cacatgacct tacctatcat tggagatgac tcacactctt taccctgccc cttttgcttt 180 gtatccaata aataacagca cagccagaca ttcggggcca ctaccagtct ccgcgcattg 240 ctggtagtgg tcccccgggc ccagctgtct tttcttttat ctcttcgtct tgtgtcttta 300 tttctacact ctctcgtcgc cgcacacagg gagagaccca ctgaccctgt ggggctggtc 360 cctacagtaa ttttaaaggg aagagcaaca aactttcggt ttgcagggct gggactgttt 420 acagctgcaa aatttagaga ggacatcaat ctattattat ccacatttta cagctgggga 480 aatcaatgct aagagaggaa attcatttgc ccagaggtgc accaccctgg cctccaatgt 540 gcaattcatg caattgtgat ttccgacctg gtcccaaact aaccctaaag ttagcaggcc 600 agaacagtgc tgctcaaata agtcagctta gtcaaataag tcaggcaaag gtcgtgtctt 660 tgcacctgga gtcctggcca ggctggtagg tccctcctcc tgggacaagt tcaccctcag 720 aattttcagc aagatcatct cccacagctt gttaattggt tcttggttct aagtgatttt 780 tttgtttatt ggtttaagag atgggatccc actctatcac ccaggcttga gtgccgtggc 840 acaatcatag ctcgctgcag cctcaaactc ctgggctcga gtgatcctcc tgcctcagcc 900 tcccagcctc agcctgggac cacaggcatg taccaccatg cctggctcta agtggcttta 960 atggggtcct tctgagggat gttggagtca gggcctgggg ggagttcccc aggccttctg 1020 ggaggcctgg gctctggact tgacctcgcc tactgtctgg ccctgctgaa aagaaaaaaa 1080 aacatggaaa tggcagacct aacagaatct gggctgtggt caggatgtgg ctgaagaagc 1140 cacaagaaaa acatgcagtc ccctttcagc ggtcatgccc agcagttggg tgccgataat 1200 gggcctgatt tcctgtagga agccctggct ctcttggcca catggacagt gtctgaggct 1260 ggccctgtta ttcccctttg cagatgaaga aacaggctca gagagtttac ctggtatcct 1320 ggagtcccag gagcactttt tctggaagta ggagcttgtt tcctgcaggt gccaagacag 1380 agaccgacat tgtttgttgg ctgggtcggt ctcccagttt tcagctggct ccagtctcac 1440 ctgttgctca cacaccctcc atgtctccca tagtcccctc ggtggggaca gaggcactgg 1500 atgaagccct gctcgtcacc acagagacac ctgaacacaa aaaccagtcc ctggggtcag 1560 acccaggccc cgcccccaga cccaggccct gccctcactc caccacgcaa ctgtgcaacc 1620 tcagtttccc caggtggaga ccggaccaac aatgatggcc tctgcctctt caggtcatag 1680 tacagatgaa tacaggctgg cacggcctag gcactcagta acacacggca gaggcacagg 1740 gacttaagat ggagtgtccc aggcagccac agttggctgg cacccagttg ggaagggccc 1800 aagggctttt aaagcagggt gaaaaaaaaa gcccacctcc tttctgggaa actgaaactg 1860 aaaacctaat taatcctctg cctgtaggtg cctcatgcaa gagctgctgg tcagagcact 1920 tcctggaact tgctattggt caggacgttt cctatgctaa taaaggggtg gcccgtagaa 1980 gattccagca ccctccccta actccaggcc agactccttt cagctaaagg ggagatctgg 2040 atggcatcta cttcgtatga c 2061 5 22 DNA Artificial Sequence Description of Artificial Sequence Primer 5 gtaatacgac tcactatagg gc 22 6 27 DNA Artificial Sequence Description of Artificial Sequence Primer 6 atccccgtct tccatgggca ctctgca 27 7 19 DNA Artificial Sequence Description of Artificial Sequence Primer 7 actatagggc acgcgtggt 19 8 28 DNA Artificial Sequence Description of Artificial Sequence Primer 8 atagtcatac gaagtagatg ccatccag 28 9 19 DNA Artificial Sequence Description of Artificial Sequence Primer 9 actatagggc acgcgtggt 19 10 25 DNA Artificial Sequence Description of Artificial Sequence Primer 10 cctcgctgtt ggccttgatg gtgaa 25 11 18 DNA Artificial Sequence Description of Artificial Sequence Primer 11 gccaacagcg aggcctgc 18 12 18 DNA Artificial Sequence Description of Artificial Sequence Primer 12 gcatccaggg aagccatt 18 13 18 DNA Artificial Sequence Description of Artificial Sequence Primer 13 actccccagg ccaaaacc 18 14 22 DNA Artificial Sequence Description of Artificial Sequence Primer 14 cgcgtcctga agcttatggt tt 22 15 18 DNA Artificial Sequence Description of Artificial Sequence Primer 15 gcgtctgcag cggtggag 18 16 18 DNA Artificial Sequence Description of Artificial Sequence Primer 16 cgaaaagccg agcaggac 18 17 21 DNA Artificial Sequence Description of Artificial Sequence Primer 17 atggcatcta cttcgtatga c 21 18 18 DNA Artificial Sequence Description of Artificial Sequence Primer 18 aaccgtgttg ccccatga 18 19 471 DNA Mus sp. CDS (1)..(468) 19 atg gca tct act tcg tat gac tat tgc aga gtg ccc atg gaa gac ggg 48 Met Ala Ser Thr Ser Tyr Asp Tyr Cys Arg Val Pro Met Glu Asp Gly 1 5 10 15 gat aag cgc tgt aag ctt ctg ctg ggg ata gga att ctg gtg ctc ctg 96 Asp Lys Arg Cys Lys Leu Leu Leu Gly Ile Gly Ile Leu Val Leu Leu 20 25 30 atc atc gtg att ctg ggg gtg ccc ttg att atc ttc acc atc aag gcc 144 Ile Ile Val Ile Leu Gly Val Pro Leu Ile Ile Phe Thr Ile Lys Ala 35 40 45 aac agc gag gcc tgc cgg gac ggc ctt cgg gca gtg atg gag tgt cgc 192 Asn Ser Glu Ala Cys Arg Asp Gly Leu Arg Ala Val Met Glu Cys Arg 50 55 60 aat gtc acc cat ctc ctg caa caa gag ctg acc gag gcc cag aag ggc 240 Asn Val Thr His Leu Leu Gln Gln Glu Leu Thr Glu Ala Gln Lys Gly 65 70 75 80 ttt cag gat gtg gag gcc cag gcc gcc acg tgc aac cac act gtg aag 288 Phe Gln Asp Val Glu Ala Gln Ala Ala Thr Cys Asn His Thr Val Lys 85 90 95 aga aaa cca ggt ctt aag cgt gag aat cgc gga caa gaa gta cta ccc 336 Arg Lys Pro Gly Leu Lys Arg Glu Asn Arg Gly Gln Glu Val Leu Pro 100 105 110 cag ctc cca gga ctc cag ctc cgc tgc ggc gcc cca gct gct gat tgt 384 Gln Leu Pro Gly Leu Gln Leu Arg Cys Gly Ala Pro Ala Ala Asp Cys 115 120 125 gct gct ggg cct cag cgc tct gct gca gtg aga tcc cag gaa gct ggc 432 Ala Ala Gly Pro Gln Arg Ser Ala Ala Val Arg Ser Gln Glu Ala Gly 130 135 140 aca tct tgg aag gtc cgt cct gct cgg ctt ttc gct tga 471 Thr Ser Trp Lys Val Arg Pro Ala Arg Leu Phe Ala 145 150 155 20 156 PRT Mus sp. 20 Met Ala Ser Thr Ser Tyr Asp Tyr Cys Arg Val Pro Met Glu Asp Gly 1 5 10 15 Asp Lys Arg Cys Lys Leu Leu Leu Gly Ile Gly Ile Leu Val Leu Leu 20 25 30 Ile Ile Val Ile Leu Gly Val Pro Leu Ile Ile Phe Thr Ile Lys Ala 35 40 45 Asn Ser Glu Ala Cys Arg Asp Gly Leu Arg Ala Val Met Glu Cys Arg 50 55 60 Asn Val Thr His Leu Leu Gln Gln Glu Leu Thr Glu Ala Gln Lys Gly 65 70 75 80 Phe Gln Asp Val Glu Ala Gln Ala Ala Thr Cys Asn His Thr Val Lys 85 90 95 Arg Lys Pro Gly Leu Lys Arg Glu Asn Arg Gly Gln Glu Val Leu Pro 100 105 110 Gln Leu Pro Gly Leu Gln Leu Arg Cys Gly Ala Pro Ala Ala Asp Cys 115 120 125 Ala Ala Gly Pro Gln Arg Ser Ala Ala Val Arg Ser Gln Glu Ala Gly 130 135 140 Thr Ser Trp Lys Val Arg Pro Ala Arg Leu Phe Ala 145 150 155 21 7 PRT Mus sp. 21 Met Ala Ser Thr Ser Tyr Asp 1 5 22 18 DNA Artificial Sequence Description of Artificial Sequence Primer 22 tccatgtccc ctcggtgg 18 23 28 DNA Artificial Sequence Description of Artificial Sequence Primer 23 atagtcatac gaagtagatg ccatccag 28 24 28 DNA Artificial Sequence Description of Artificial Sequence Primer 24 aaaggtacca gctgtctttc tgtctgtc 28 25 18 DNA Artificial Sequence Description of Artificial Sequence Primer 25 gtgctctccc cgctaacc 18 26 18 DNA Artificial Sequence Description of Artificial Sequence Primer 26 ggacgtttcc tatgctaa 18 27 35 DNA Artificial Sequence Description of Artificial Sequence Primer 27 aaagcggccg ctcatcactg cagcagagcg ctgag 35 28 18 DNA Artificial Sequence Description of Artificial Sequence Primer 28 aggggaactc accagacc 18 29 18 DNA Artificial Sequence Description of Artificial Sequence Primer 29 atggccctaa tggcttcc 18 30 21 DNA Artificial Sequence Description of Artificial Sequence Primer 30 cattaaacca taagcttcag g 21 31 18 DNA Artificial Sequence Description of Artificial Sequence Primer 31 ccctcaagct cctccact 18 32 29 DNA Artificial Sequence Description of Artificial Sequence Primer 32 gacggatcct aaagcttaca gcgcttatc 29 33 2456 DNA Mus sp. 33 tcccatagtc ccctcggtgg ggacagaggc actggatgaa gccctgctcg tcaccacaga 60 gacacctgaa cacaaaaacc agtccctggg gtcagaccca ggccccgccc ccagacccag 120 gccctgccct cactccacca cgcaactgtg caacctcagt ttccccaggt ggagaccgga 180 ccaacaatga tggcctctgc ctcttcaggt catagtacag atgaatacag gctggcacgg 240 cctaggcact cagtaacaca cggcagaggc acagggactt aagatggagt gtcccaggca 300 gccacagttg gctggcaccc agttgggaag ggcccaaggg cttttaaagc agggtgaaaa 360 aaaaagccca cctcctttct gggaaactga aactgaaaac ctaattaatc ctctgcctgt 420 aggtgcctca tgcaagagct gctggtcaga gcacttcctg gaacttgcta ttggtcagga 480 cgtttcctat gctaataaag gggtggcccg tagaagattc cagcaccctc ccctaactcc 540 aggccagact cctttcagct aaaggggaga tctggatggc atctacttcg tatgactatt 600 gcagagtgcc catggaagac ggggataagc gctgtaagct tctgctgggg ataggaattc 660 tggtgctcct gatcatcgtg attctggggg tgcccttgat tatcttcacc atcaaggcca 720 acagcgaggc ctgccgggac ggccttcggg cagtgatgga gtgtcgcaat gtcacccatc 780 tcctgcaaca agagctgacc gaggcccaga agggctttca ggatgtggag gcccaggccg 840 ccacctgcaa ccacactgtg gtaagctcct caactccttt ggatggccta gtactaggcg 900 gtgggaggga caagaatctc tccccagaaa tctgacccag ggtgggtctc cagggagatg 960 caggggaggt cctgaaactg ctcctgggcc cccacatcaa gggacctagg ttcccctacc 1020 agggtttgtg ggcccctaac ccagtccagg gcactggtgt aggggcaggg tgttaaaact 1080 ctccagatcc cccaaatcgg ggacctcagt atccccctgg gacttaggtg aatttataaa 1140 ttctttccag ggcactggtg tcgggggcct tgaaactcct cgtgggcacc agtcctgggg 1200 gagtagaaat ccctattcag ggttgaaggg ggacctcacc agaccctgaa aaagggggct 1260 tttgaaattt tcacttcatc cctaagaaac tgaaatattc acctgggtcc tgatatgggg 1320 gatcttgaaa ctctcgctgg gcatgtcact tgggcgggga aatcccactg cattctggat 1380 ttggtagggc cctctaactt ttcttgggcc attgctcagg caatctggaa atgtccacta 1440 aactttggtt atcgatagcc tccaagtttc cacgtggggt ggcctcaaaa ctcccatttt 1500 gaggacccac atgcttatgg gtggccctgg gagagtgtgt ggttgtggct gttctttaag 1560 gttggagacc atggtgcaga gagggttgga agaaaacctg aaaggggttt gcatttaagc 1620 ccctctgtcc ccaggaccta gggaggaggc ccaggtccca ggggcagcag ccaaactccc 1680 caggccaaaa ccccagattc taactcttct tagatggccc taatggcttc cctggatgca 1740 gagaaggccc aaggacaaaa gaaagtggag gagcttgagg gtgagaaagg gagaagggag 1800 agggccgggg aggggtgagt caggtatgga agagggggtg ggggcaggga gaccagggct 1860 ggaggttggg gtaaggggga ggttctgtcc cagagtggag cagggcccca gcatggccac 1920 atgctgaccc gccccctgtt tctgtcctcc caccctacca ggagagatca ctacattaaa 1980 ccataagctt caggacgcgt ctgcagaggt ggagcgactg aggtcagaga tagccttccc 2040 ccgctaccct ccacctgcca cattcctctc acccccacat ccctagccca agacccagga 2100 tctcctttgc tcccaaaatc cccattgccc caagggataa agtttgagtc ccacaaaagg 2160 ataacttagc ccctagggtc acagagccat gggtggccgc tgtccattcc ctccccggtg 2220 acttggattg gggcggtgcg gggggaactc ccgggggcgg tgggcttaca gggagggcgg 2280 caggagccag gacgagcaga tgcctgattt gcccccatct gtaccgcaga agagaaaacc 2340 aggtcttaag cgtgagaatc gcggacaaga agtactaccc cagctcccag gactccagct 2400 ccgctgcggc gccccagctg ctgattgtgc tgctgggcct cagcgctctg ctgcag 2456 

What is claimed is:
 1. An isolated genomic DNA encoding HM1.24 antigen protein, said DNA containing 4 axon regions encoding the amino acid sequence as set forth in SEQ ID NO:
 2. 2. The genomic DNA according to claim 1, comprising 4 exons and 3 introns and encoding the amino acid sequence as set forth in SEQ ID NO:
 2. 3. An isolated splicing variant of the genomic DNA as set forth in claim 1, wherein the variant encodes an HM1.24 antigen protein that differs from SEQ ID NO: 2 by lacking at least one of the exons 1 to
 4. 4. An isolated splicing variant of the DNA as set forth in claim 1, wherein the variant encodes an HM1.24 antigen protein that differs from SEQ ID NO: 2 by lacking exon
 2. 5. An isolated splicing variant of the DNA as set forth in claim 1, wherein the variant encodes an HM1.24 antigen protein that differs from SEQ ID NO: 2 by lacking exons 2 and
 3. 6. A process for producing HM1.24 antigen protein which method comprises culturing animal cells transformed with an expression vector comprising the genomic DNA according to claim
 1. 